Defects in cardiac excitability are the basis for human arrhythmia and sudden cardiac death, a leading cause of mortality in developed countries. Recent findings demonstrate a new paradigm for human arrhythmia based on gene mutations that affect the expression/subcellular localization of cardiac ion channels and transporters. Human type 4 long QT syndrome (LQT4) results from loss-of-function mutations in the membrane adapter ankyrin-B (AnkB). Subjects with LQT4, and mice with reduced AnkB expression display similar complex cardiac phenotypes including atrial, ventricular, conduction defects, and risk of sudden cardiac death. However, the molecular identities of AnkB polypeptides, scope of AnkB expression in specialized cardiac cells, and cellular role(s) for AnkB polypeptides for cardiac excitability remain critical, yet unanswered questions. Moreover, the mechanisms underlying AnkB regulation in normal heart, and dysfunction in human arrhythmia remain unsolved. The long-term objective of this research proposal is to understand the molecular basis for AnkB function in the heart. We hypothesize that coordinate dysfunction of AnkB polypeptides throughout the heart create the complex phenotype of human type 4 long QT syndrome due to defects in ion channel/transporter trafficking and membrane stability. The specific aims are to: 1) Characterize the expression and subcellular distribution of AnkB isoforms in diverse excitable cell types of heart. 2) Define the cellular role(s) of AnkB for ion channel/transporter trafficking and localization in adult cardiomyocytes using recently developed lentiviral techniques. 3) Characterize the mechanisms underlying AnkB regulation in heart, and determine how human AnkB loss-of-function mutations associated with fatal arrhythmia affect this regulation. The cellular pathways underlying ion channel and transporter targeting, localization, and stability in cardiomyocytes are essentially unknown but present an exciting new target for future cardiac therapies. We propose to use recently developed expression techniques to elucidate the molecular mechanisms underlying AnkB-dependent cellular pathways for ion channel and transporter targeting, localization, and stability in the physiological context of the primary cardiomyocyte. It is anticipated that this information will advance understanding of mechanisms underlying AnkB-based human fatal human arrhythmia as well as acquired cardiac arrhythmias associated with abnormal Ca2+ homeostasis, and begin to define potential future molecular targets for the regulation of cellular excitability.